Rat embryonic stem cells create new era in development of genetically manipulated rat models.

Embryonic stem (ES) cells are isolated from the inner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer gene-modified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.

miR-148a plays a pivotal role in the liver by promoting the hepatospecific phenotype and suppressing the invasiveness of transformed cells.

UNLABELLED: MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post-transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR-148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR-148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the "adult liver" phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR-148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR-148a expression was frequently down-regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR-148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR-148a silencing had the opposite consequences. Finally, we showed that miR-148a exerted its tumor-suppressive effect by regulating the c-Met oncogene, regardless of the DNMT1 expression level. CONCLUSION: miR-148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme.

Kinetic model to predict the absorption of nasally applied drugs from in vitro transcellular permeability of drugs.

The purpose of this study is to propose a kinetic model to predict the absorption of nasally applied drugs from their permeability to the Caco-2 monolayer (P(Caco-2)). Since a drug applied to the nose in an in vivo physiologic condition is translocated to the gastrointestinal (GI) tract by coordinated beats of cilia (mucociliary clearance, MC), the drug undergoes absorption both from the nasal cavity and from the GI tract. The detailed MC of the rat was examined, using inulin as a marker of the applied solution. Inulin disappeared monoexponentially from the nasal cavity, indicating that the MC can be assumed to follow first-order kinetics. From the disappearance of inulin, the first order rate constant for MC (k(MC)) was calculated as 0.0145 min(-1). In the proposed kinetic model, the fractional absorption of the drug following nasal application is predicted as the sum of F(NC) (fractional absorption from the nasal cavity) and F(GI) (fractional absorption from the GI tract), both of which are estimated indirectly from P(Caco-2). F(NC) is calculated according to the equation, k(a)/(k(a)+k(MC)), where k(a) is the absorption rate constant. Nasal drug absorption is assumed to follow first order kinetics. The k(a) of four drugs was initially calculated from k(MC) and their F(NC); thereafter, the linear relationship between k(a) and P(Caco-2), from which k(a) is predicted, was determined. F(GI) is calculated as F(p.o.)(1-F(NC)), where F(p.o.) is fractional absorption after oral administration. F(p.o.) was predicted from the previously determined sigmoid curve between F(p.o.) and P(Caco-2). The proposed kinetic model is the first estimation system for nasal drug absorption based on drug disposition after nasal application and is useful for the development of nasal dosage forms.

Evaluation of the contribution of the nasal cavity and gastrointestinal tract to drug absorption following nasal application to rats.

Drugs applied to the nose in in vivo physiologic condition undergo absorption from the nasal cavity and the gastrointestinal (GI) tract because drug solution in the nasal cavity, together with mucus layer, is cleared to pharynx and then to the GI tract by coordinated beat of the cilia on nasal epithelial cells. The purpose of this study was to develop evaluate the contribution of the nasal cavity and the GI tract to drug absorption following nasal application and to clarify the relation to the transepithelial permeability of the drug (the permeability to Caco-2 monolayer, P(Caco-2)). Male Wistar rats received intravenous, nasal, and oral drug administration and drug concentration-time profiles in plasma were determined. Fractional absorption after nasal application (Fn) and oral administration (Fpo) were calculated from the area under the curve following intravenous injection (AUCiv), nasal application (AUCn), and oral administration (AUCpo) as AUCn/AUCiv and AUCpo/AUCiv, respectively. Fractional absorption from the nasal cavity (F(NC)) and the GI tract (F(GI)) following nasal application was calculated as (Fn-Fpo)/(1-Fpo) and Fpo(1-F(NC)), respectively. The shape of the curve between F(NC) and P(Caco-2) was similar with the one observed in the case of oral bioavailability except the curve shifted right. It is noteworthy that the relation between F(GI) and P(Caco-2) showed a bell-shaped curve with peak at 10(-6) cm/s of P(Caco-2). Highly permeable drug is primarily absorbed through the nasal mucosa before it is cleared to the GI tract. With the decrease in P(Caco-2), the larger amount of the drug is cleared to the GI tract and absorption from the GI tract is increased. Poorly permeable drug, on the other hand, was absorbed neither from the nasal was nor the GI tract. These findings suggest that the primary absorption site of drug after nasal application is decided by mucociliary clearance and absorption through the nasal mucosa.